Indian Journal of Microbiology Research 2020;7(2):212–217

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Indian Journal of Microbiology Research

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Original Research Article

Molecular characterization of metallo-beta-lactamase producers amongcarbapenem resistant Pseudomonas aeruginosa isolated from cases of hospitalacquired infections in a tertiary care hospital, Bengaluru

Ashwini Sondakar1, Sneha K Chunchanur2, Ambica Rangaiah2,Sathyanarayan Muthur Shankar2,*1Dept. of Microbiology, Belgaum Institute Medical Sciences, Belagavi, Karnataka, India2Dept. of Microbiology, Bangalore Medical College & Research Institute, Bengaluru, Karnataka, India

A R T I C L E I N F O

Article history:Received 04-02-2020Accepted 13-03-2020Available online 20-07-2020

Keywords:MetallobetalactamasesCarbapenemasePseudomonas aeruginosa

A B S T R A C T

Background: Pseudomonas aeruginosa is an important cause of multidrug-resistant nosocomial infections.The knowledge of resistance mechanisms in Pseudomonas is an important issue for antimicrobial treatment.Therefore, the objective was to detect carbapenems resistance in P. aeruginosa by phenotypic methods andgenes (blaIMP and blaVIM) coding for carbapenemase resistance.Materials and Methods: The study conducted in the Department of Microbiology, BMCRI, Bengaluru.91 samples from the patients hospitalised for 48 hours and more were processed. Carbapenem resistantP. aeruginosa (CRPA) were identified by biochemical tests and by Kirby Bauer Disk diffusion method asper CLSI guidelines. Those carbapenem resistant isolates were further subjected to two MBL detectingphenotypic tests- Modified Hodge Test (MHT) and Combined disk Test (CDT) by using imipenem andimipenem/ EDTA disk and MBL genes (blaIMP and blaVIM) were identified by PCR.Results: 91 clinical isolates were identified as CRPA, 92.3% isolates were positive by CDT whereas only13.2% of isolates showed positive by MHT method indicating MBL production. Among 91 strains, 19.04%strains were harbouring blaVIM and 1.2% strain harbouring blaIMP gene.Conclusion: The detection of MBL-producing P. aeruginosa help in appropriate antimicrobial therapy andavoid development and dissemination of these strains. Hence routine detection of MBL production in P.aeruginosa should be undertaken. All CRPA isolates should be routinely screened for MBL productionusing CDT and positive isolates should be confirmed by PCR. MHT test had low sensitivity. To understandthe epidemiology, there is a need of genetic analysis and typing of MBL enzymes.

© 2020 Published by Innovative Publication. This is an open access article under the CC BY-NC license(https://creativecommons.org/licenses/by-nc/4.0/)

1. Introduction

Pseudomonas aeruginosa is a Gram-negative bacteriumwith the ability to persist in both community andhospital settings. It is an important nosocomial pathogenwith emerging resistance to many effective groups ofantibiotics through both intrinsic and acquired resistancemechanisms. Carbapenems are the drug of choice forinfections caused by Pseudomonas aeruginosa includingother Gram-negative organisms.1Global emergence of

* Corresponding author.E-mail address: drsathyams@gmail.com (S. M. Shankar).

carbapenem resistance Pseudomonas aeruginosa (CRPA)including resistance to beta-lactams, Aminoglycosides,and Fluoroquinolones represents an extraordinary threatto public health. The major mechanisms of carbapenemresistance in Pseudomonas aeruginosa include carbapenemhydrolyzing enzymes i.e. carbapenemase, decrease outermembrane permeability and alteration penicillin-bindingproteins.2 However, the clinical use of these antimicrobialsis under threat with the emergence of carbapenemases,particularly Metallo- ß-lactamases (MBLs).2 MBL belongsto Ambler class B, and these enzymes can hydrolyze awide variety of beta-lactam agents, such as penicillin,

https://doi.org/10.18231/j.ijmr.2020.0382394-546X/© 2020 Innovative Publication, All rights reserved. 212

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cephalosporins, and carbapenems. They require zinc fortheir catalytic activity and are inhibited by metal chelators,such as EDTA and thiol-based compounds.3 MBLs in P.aeruginosa were identified in 1991 in Japan4 and sincethen have been described from various parts of the world,including Asia, Europe, Australia, South America, andNorth America4,5whereas in India blaV IM , blaIMP andblaNDM genes are frequently encountered in P. aeruginosa.6

The genes responsible for the production of MBLsare typically part of an integron structure and are carriedon transferrable plasmids but can also be part of thechromosome.7 Due to integron-associated gene cassettes,MBL producing P. aeruginosa isolates are often resistantto different groups of antimicrobial agents, which can betransferred to another Gram-negative bacteria.8

MBLs in Carbapenem-resistant Pseudomonas aerugi-nosa can be detected by different phenotypic methods andthese methods are based on the ability of metal chelators toinhibit the activity of MBLs such as EDTA and thiol-basedcompounds.9 These include Combined Disk Test (CDT)9

using EDTA with imipenem (IPM), Modified Hodge test(MHT), MBL Epsilometer test (E-test) and EDTA diskpotentiation test.10,11 A PCR detection assay is consideredas a gold standard method for the detection of MBLproducers.12 Because of the increasing rate of resistance tothe carbapenems, the treatment of infections produced byMBLs producing P. aeruginosa is becoming critical.

Hence the present study was undertaken to detect pres-ence of MBLs among Carbapenem-Resistant Pseudomonasaeruginosa (CRPA) isolates by phenotypic methods likeMHT and Combined Disk Test (CDT) by using Imipenem(IPM)- Ethylene diamine tetraacetic acid (EDTA) and toidentify the MBL genes (blaVIM and blaIMP) coding forcarbapenemase resistance by conventional PCR in a tertiarycare hospital in Bengaluru.

2. Materials and Methods

This study was conducted over a period of one year fromJanuary 2018 to December 2018 at a tertiary hospital,BMCRI, Bengaluru. Ethical clearance was obtained fromthe institutional ethical committee. This study included 91non-consecutive clinical samples from patients hospitalizedfor 48 hours and more, received for culture and sensitivityin the Department of Microbiology.

All the clinical specimens were subjected to directmicroscopy, growth on culture media and series of testsfor identification of P. aeruginosa. These isolates weresubjected to antimicrobial susceptibility testing by Kirby-Bauer disk diffusion method according to CLSI guide-lines.13 The antibiotics tested include amikacin (30µg),ciprofloxacin (5µg), ceftazidime (30µg), piperacillin-tazobactam (100/10µg), imipenem (10µg), meropenem(MEM-10µg), aztreonam (30 µg), colistin (10 µg) andpolymyxin-B (300 units). All the disks were obtained

commercially (Hi-Media Laboratories Limited. Mumbai,India). ATCC strain of Pseudomonas aeruginosa 27853 isused as control.

All isolates resistant to imipenem or meropenem orceftazidime or any two of them were considered as probableMBL producer. All positive isolates were further tested bytwo phenotypic tests for the MBL detection, described asfollows.

2.1. Imipenem- Ethylene diamine tetraacetic acidcombined disc test

A lawn culture of test isolate was prepared. Allowed to dryfor five minutes. Two imipenem (10 µg) discs, one with 0.5M EDTA and other a plain imipenem disc, were placed onthe surface of agar plates approximately 30mm apart. Theplates were incubated overnight at 37oC for 16-18h. Anincrease in zone diameter of >7mm around the imipenem-EDTA disc in comparison to imipenem disk alone indicatesthe production of MBL.9

2.2. Modified Hodge Test (MHT)

A saline suspension of a 0.5 McFarland standard of E.coli ATCC 25922 was prepared and diluted 1:10 and lawninoculated on Muller Hinton Agar (MHA). The plate wasallowed to dry for 3-10 minutes. An imipenem (10 µg) diskwas placed at the center and 3-5 colonies of test organismswere inoculated in a straight line drawn out from the edge ofthe disk. A known NDM positive strain was used as Positivecontrol and incubated overnight at 350C for 20-24h. thepresence of a distorted zone of inhibition or cloverleaf typeof indentation at the intersection of the test organism andE. coli. within the zone of inhibition of the IPM disk wasinterpreted as a positive result.10,11

2.3. PCR for carbapenem encoding gene

Isolates tested positive in the phenotypic test are subjectedto conventional PCR for detection genes coding for MBLs.The DNA extraction was done by the crude method (boilingmethod). Freshly subcultured colonies were suspended in 50µl of PCR grade water, heated to 990C for 10 minutes in awater bath and kept at room temperature for 5 minutes. Thesuspension is centrifuged at 14000 rpm for 1min at 40C, 5µl of supernatant was used as the template for a 50 µ l PCRreaction. PCR was performed by using previously designedprimers (Sigma-Aldrich, Bengaluru) for blaV IM , and blaIMP(Table 1)14

Amplification was performed in 50 µ l PCR mixtureconsisting of master mix (25 µl)- Taq DNA polymerase2x master mix RED 1.5 mM MgCl2 (Synergy ScientificService PVT. LTD), PCR grade water (18µ l), PrimerblaV IM , and blaIMP F’ and R’ (1µl each) and DNA (5µl).

DNA was amplified in a Master cycler Eppendorf underthe following conditions (Table 1) and PCR products

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were kept at 40C. Known blaV IM , and blaIMP, producinglaboratory strain Pseudomonas aeruginosa was used as thepositive control. Pseudomonas aeruginosa ATCC 27853reference strain was used as the MBLgene’s negative strain.Cycling conditions for MBL (blaVIM and blaIMP) genewere described (Table 2).12

The PCR products were analyzed by gel electrophoresiswith 2% agarose gel in TAE (tris-acetate buffer) bufferwith1.5µl ethidium bromide and were visualized andphotographed under ultraviolet illumination.

3. Results

Among ninety-one CRPA isolates included in the study70.3% were from the pus, 12.08% from blood and bodyfluids, 6.5% from urine, 3.3% from respiratory samplesand 3.29% from others (Corneal and ENT). Distributionof isolates according to wards as follows, General surgery(42.8%), burns and plastic surgery (27.4%), ICUs (9.8%),orthopedics (4.3%), Nephro-urology (4.3%) and others -ENT and ophthalmic (2.19%).

The antimicrobial susceptibility testing of CRPAisolates showed resistance to other antimicrobials includingceftazidime and cefepime(95.6% each), ciprofloxacinand levofloxacin (91.2% each), piperacillin(94.5%),ticarcillin (96.7%), piperacillin-tazobactam(86.8%),amikacin(83.5%), gentamicin(86.8%), aztreonam(80.2%),whereas only 12.08% isolates were resistant to colistin(Figure 1).

Fig. 1: Antimicrobial susceptibility test in Carbapenem resistantpseudomonas aeruginosa (CRPA) isolates. Antimicrobial sus-ceptibility test in carbapenem resistant pseudomonas aeruginosa(CRPA) isolates

3.1. MBL phenotypic screening tests

Among the two phenotypic tests, Combined Disc Testusing imipenem and EDTA was positive in 84 isolates andModified Hodge Test was positive in only 12 isolates. In thisstudy, CDT showed high sensitivity (92.3%) while MHTwas least sensitive (13.18%) (Figure 2).

Fig. 2: MBL phenotypic screening tests

3.2. PCR for carbapenem encoding gene

Positively tested isolates by MBL phenotypic screeningtests are further confirmed by conventional PCR. Out of 84isolates, 16 isolates carried blaV IM (19.04%) and one isolatecarried blaIMP gene (1.19%). (Figures 3, 4 and 5)

Fig. 3: MBL genes coding for Carbapenen resistance

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Table 1: Primer sequence used in PCR and their product size as follows. 14

Primer Primer sequence (5’-3’) Product size (base pairs)bla VIM-F TTT GGT CGC ATA TCG CAACG 500bla VIM-R CCA TTC AGC CAG ATC GGC AT 500bla IMP-F GTT TAT GTT CAT ACW TCG 432bla IMP-R GGT TTA AYA AAA CAA CCA C (W=A or T; Y=C or T) 432

Table 2: Cycling conditions for different gene identification 12

Steps in PCR VIM IMPInitial DNA denaturation 940C 5min 940C 5minFinal DNA denaturation 950C 30sec 950C 30secPrimer annealing 660C 1min 450C 1minPrimer extension 720C 30 sec 720C 26 secFinal extension or Holding temperature 720C 10 min 720C10 minCycles 30 30

Fig. 4: Agarose gel run for blaVIM

4. Discussion

Antimicrobial resistance is a significant route causeof healthcare-associated infections all over the world.Carbapenem has been used as the last choice of treatmentof many Gram-negative organisms. In the current study, wehave included carbapenem-resistant P. aeruginosa isolates.Carbapenem resistance among Gram-negative bacteria has

Fig. 5: Agarose gel run for blaIMP

been increased in recent years in the Indian subcontinentand MBL producing isolates have emerged worldwide andare associated with outbreaks in health care settings over thepast few years.

In this study, most of the carbapenem-resistant Pseu-domonas aeruginosa isolates were from pus (70.3%)and from general surgery ward (42.8%) and mostof them were resistant to other class of antibiotics

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such as Anti-pseudomonal Penicillin’s, Aminoglycosides,Fluoroquinolones, Cephalosporins, and Aztreonam. Thisindicates the concomitant presence of other beta-lactamases.Polymyxins showed susceptibility of 87.9% (n=91) whichwas high compared to other classes of antibiotics and henceconsidered as a treatment option of CRPA isolates.

In this study, MBL production was screened by theModified Hodge test and Combined Disk Test by usingImipenem-EDTA. Out of 91 isolates, MHT was positive in84 isolates (92.3%) and MHT was positive in 12 isolates(13.18%). PCR detected MBL genes blaV IM/blaIMP in20.2% (blaV IM-16 and blaIMP-1) among those positiveisolates by phenotypic tests. This coincides with a highprevalence of blaV IM among Pseudomonas isolates, a studyby Amudhan M et al.14 The remaining 71 isolates that wereMBL phenotypic test positive, were negative for both MBLgenes (blaVIM/blaIMP) suggesting the presence of other MBLgenes such as SIM-1, GIM-1, NDM-1 or SPM-1.

Despite the good performance of inhibitor-basedmethods (Combined disk test using IMP-EDTA) for thedetection of MBL, false-positive results were found, hencethis is not considered as a specific test. False positives maybe due to EDTA which acts on the membrane of a bacterialcell and increases cell permeability. MHT also showed verylow sensitivity. Hence the results of the MBL phenotypictests must be interpreted cautiously.

The overall blaV IM/blaIMP production among studyisolate was 20.2% among them, blaV IM was found in 16isolates whereas blaIMPwas found in one isolate. In AsiaMBL genes, blaVIM and blaIMP are prevalent and blaIMP isfound in Japan, Korea, China, Taiwan and Iran.15–17

The prevalence of MBL genes in India ranges from 7-65% among P. aeruginosa. In a study from India showed, therate of MBL production was 24.5% among 61 P. aeruginosaisolates and blaV IM was the most common. Another studyfrom India also reported blaVIM -2 from P. aeruginosa.18

In a nationwide survey conducted to characterize 301 MBLproducing Pseudomonas species in 10 medical centers fromIndia, the MBL genes were detected in 18.9% isolates.19

There are fewer data available on the prevalence anddistribution of MBL among Indian isolates.

In this study, MBL phenotypic tests were positive in92.3% isolates and among them, 20.2% isolates werecarrying blaV IM/blaIMP genes indicating carbapenemaseproduction. So, early detection of MBL production inCarbapenem-resistant Pseudomonas aeruginosa will notonly help in treating the infections caused by them ade-quately and also help in preventing the spread of multidrugresistance to other Gram-negative strains. Therefore, allclinical isolates that are resistant to carbapenem must bescreened for MBL production by using simple phenotypictests and confirmed by the MBL Epsiolometer test (E-test)or by PCR if possible.

To conclude, carbapenem resistance in P. aeruginosais chiefly mediated by Metallo-beta-lactamase production.

Among the two phenotypic tests performed in this study,CDT was more sensitive compared to the Modified HodgeTest in the screening of MBL production. Genotypically,the common MBL gene -found was blaVIM comparedto blaIMP. Therefore, screening for MBL production inmicrobiology laboratories is crucial for optimal treatmentof patients, particularly hospitalized patients and alsoto prevent the possible spread of resistance to otherGram-negative organisms because of their broad-spectrumdrug resistance which creates a therapeutic challenge toclinicians. Finally, to understand the epidemiology, there isa need for genetic analysis and also typing of Metallo-ß-lactamase enzymes.

5. Source of Funding

None.

6. Conflict of Interest

None.

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Author biography

Ashwini Sondakar Senior Resident

Sneha K Chunchanur Assistant Professor

Ambica Rangaiah Professor and Head

Sathyanarayan Muthur Shankar Assistant Professor

Cite this article: Sondakar A, Chunchanur SK, Rangaiah A, ShankarSM. Molecular characterization of metallo-beta-lactamase producersamong carbapenem resistant Pseudomonas aeruginosa isolated fromcases of hospital acquired infections in a tertiary care hospital,Bengaluru. Indian J Microbiol Res 2020;7(2):212-217.